It remains unanswered whether and how intraoperative frozen section analysis contributes to the surgical margin status on radical prostatectomy specimens. We aimed to determine whether frozen section analysis during radical prostatectomy reduces the incidence of positive surgical margins. We retrospectively analyzed a consecutive series of patients undergoing robot-assisted laparoscopic radical prostatectomy performed at our institution between 2004 and 2011. We identified 2608 cases, including 1128 (43.3%) where intraoperative frozen section analysis was performed to assess surgical margins. Of the cases with positive (n = 60; 5.3%)/negative (n = 1029; 91.2%)/atypical or indeterminate (n = 39; 3.5%) frozen section analyses, 22 (36.7%)/83 (8.1%)/4 (10.3%) were found to have positive surgical margins on radical prostatectomy specimens, respectively. Thus, 109 (9.7%) of 1128 cases with frozen section analysis had positive surgical margins, compared with 163 (11.0%) of 1480 cases with no frozen section analysis (P = .264). When the patients were subgrouped by histopathologic characteristics, frozen section analysis led to a considerable reduction in the rate of positive surgical margins in cases with biopsy Gleason score 7 (12.4% → 8.7%; P = .087)/8 (28.6% → 16.3%; P = .048)/≥7 (15.3% → 10.1%; P = .012) tumor or pT3b (36.6% → 23.2%; P = .075)/≥pT3b (38.1% → 25.4%; P = .091) disease. Multivariate analysis further revealed that performing frozen section analysis in biopsy Gleason score 7 or higher tumors was an independent predictor of negative surgical margins (odds ratio, 0.61; P = .018). In addition, frozen section analysis of the distal urethra or apex of the prostate (7.5%, P = .035) as well as multiple negative frozen section analyses (≥2: 6.2%, P = .001; ≥4: 2.2%, P = .007) correlated with significantly lower rates of positive surgical margin, compared with no frozen section analysis. Overall, intraoperative frozen section analysis did not dramatically change surgical margin status of radical prostatectomy. Nonetheless, it could be useful in preventing incomplete tumor resection, especially in men with high-grade (Gleason score ≥7) tumor at the apex.